Human GIL-19/AE289 proteins

ABSTRACT

Novel human GIL-19/AE289 protein is disclosed which shows a high degree of homology to interleukin-10 (IL-10). Polynucleotides encoding such protein are also enclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/013,741, filed Dec. 17, 2004 now U.S. Pat. No. 7,459,533, which is adivisional of U.S. application Ser. No. 09/561,811, filed Apr. 28, 2000,now U.S. Pat. No. 7,307,161, which claims the benefit of U.S.Provisional Patent Application No. 60/131,473, filed Apr. 28, 1999. Thecontents of the above-referenced patent applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides novel proteins which show homology tointerleukin-10 (IL-10) and polynucleotides encoding such proteins, alongwith therapeutic, diagnostic and research utilities for thesepolynucleotides and proteins.

BACKGROUND OF THE INVENTION

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, CSFs and interleukins) hasmatured rapidly over the past decade. The now routine hybridizationcloning and expression cloning techniques clone novel polynucleotides“directly” in the sense that they rely on information directly relatedto the discovered protein (i.e., partial DNA/amino acid sequence of theprotein in the case of hybridization cloning; activity of the protein inthe case of expression cloning). More recent “indirect” cloningtechniques such as signal sequence cloning, which isolates DNA sequencesbased on the presence of a now well-recognized secretory leader sequencemotif, as well as various PCR-based or low stringency hybridizationcloning techniques, have advanced the state of the art by makingavailable large numbers of DNA/amino acid sequences for proteins thatare known to have biological activity by virtue of their secreted naturein the case of leader sequence cloning, or by virtue of the cell ortissue source in the case of PCR-based techniques. It is to theseproteins and the polynucleotides encoding them that the presentinvention is directed.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising an isolated polynucleotide selected from the group consistingof:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:1;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO: 1 from nucleotide 65 to nucleotide 601;    -   (c) a polynucleotide comprising the nucleotide sequence of the        full-length protein coding sequence of clone hGIL-19/AE289        deposited under accession number ATCC 207231;    -   (d) a polynucleotide encoding the full-length protein encoded by        the cDNA insert of clone hGIL-19/AE289 deposited under accession        number ATCC 207231;    -   (e) a polynucleotide comprising the nucleotide sequence of a        mature protein coding sequence of clone hGIL-19/AE289 deposited        under accession number ATCC 207231;    -   (f) a polynucleotide encoding a mature protein encoded by the        cDNA insert of clone hGIL-19/AE289 deposited under accession        number ATCC 207231;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:2;    -   (h) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:2 having biological        activity, the fragment comprising eight contiguous amino acids        of SEQ ID NO:2;    -   (i) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(f) above;    -   (j) a polynucleotide which encodes a species homologue of the        protein of (g) or (h) above;    -   (k) a polynucleotide that hybridizes under stringent conditions        to any one of the polynucleotides specified in (a)-(h); and    -   (l) a polynucleotide that hybridizes under stringent conditions        to any one of the polynucleotides specified in (a)-(h) and that        has a length that is at least 25% of the length of SEQ ID NO: 1.

Preferably, such polynucleotide comprises the nucleotide sequence of SEQID NO:1 from nucleotide 65 to nucleotide 601; the nucleotide sequence ofthe full-length protein coding sequence of clone HGIL-19/AE289 depositedunder accession number ATCC 207231; or the nucleotide sequence of amature protein coding sequence of clone hGIL-19/AE289 deposited underaccession number ATCC 207231 (e.g., nucleotides 1-1177 of SEQ ID NO: 1).In other preferred embodiments, the polynucleotide encodes thefull-length or a mature protein encoded by the cDNA insert of cloneHGIL-19/AE289 deposited under accession number ATCC 207231 (e.g., aminoacids 1-179 of SEQ ID NO: 2). In further preferred embodiments, thepresent invention provides a polynucleotide encoding a proteincomprising a fragment of the amino acid sequence of SEQ ID NO:2 havingbiological activity, the fragment preferably comprising eight (morepreferably twenty, most preferably thirty) contiguous amino acids of SEQID NO:2, or a polynucleotide encoding a protein comprising a fragment ofthe amino acid sequence of SEQ ID NO:2 having biological activity, thefragment comprising the amino acid sequence from amino acid 84 to aminoacid 93 of SEQ ID NO:2.

Other embodiments provide the gene corresponding to the cDNA sequence ofSEQ ID NO: 1.

Further embodiments of the invention provide isolated polynucleotidesproduced according to a process selected from the group consisting of:

-   -   (a) a process comprising the steps of:        -   (i) preparing one or more polynucleotide probes that            hybridize in 6×SSC at 65 degrees C. to a nucleotide sequence            selected from the group consisting of:            -   (aa) SEQ ID NO: 1, but excluding the poly(A) tail at the                3′ end of SEQ ID NO:1; and            -   (ab) the nucleotide sequence of the cDNA insert of clone                hGIL-19/AE289 deposited under accession number ATCC                207231;        -   (ii) hybridizing said probe(s) to human genomic DNA in            conditions at least as stringent as 4×SSC at 50 degrees C.;            and        -   (iii) isolating the DNA polynucleotides detected with the            probe(s);

and

-   -   (b) a process comprising the steps of:        -   (i) preparing one or more polynucleotide primers that            hybridize in 6×SSC at 65 degrees C. to a nucleotide sequence            selected from the group consisting of:            -   (ba) SEQ ID NO: 1, but excluding the poly(A) tail at the                3′ end of SEQ ID NO: 1; and        -   (bb) the nucleotide sequence of the cDNA insert of clone            hGIL-19/AE289 deposited under accession number ATCC 207231;            -   (ii) hybridizing said primer(s) to human genomic DNA in                conditions at least as stringent as 4×SSC at 50 degrees                C.;        -   (iii) amplifying human DNA sequences; and        -   (iv) isolating the polynucleotide products of step (b)(iii).

Preferably the polynucleotide isolated according to the above processcomprises a nucleotide sequence corresponding to the cDNA sequence ofSEQ ID NO: 1, and extending contiguously from a nucleotide sequencecorresponding to the 5′ end of SEQ ID NO: 1 to a nucleotide sequencecorresponding to the 3′ end of SEQ ID NO: 1, but excluding the poly(A)tail at the 3′ end of SEQ ID NO: 1. Also preferably the polynucleotideisolated according to the above process comprises a nucleotide sequencecorresponding to the cDNA sequence of SEQ ID NO: 1 from nucleotide 65 tonucleotide 601, and extending contiguously from a nucleotide sequencecorresponding to the 5′ end of said sequence of SEQ ID NO: 1 fromnucleotide 65 to nucleotide 601, to a nucleotide sequence correspondingto the 3′ end of said sequence of SEQ ID NO: 1 from nucleotide 65 tonucleotide 601.

In other embodiments, the present invention provides a compositioncomprising a protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) a fragment of the amino acid sequence of SEQ ID NO:2, the        fragment comprising eight contiguous amino acids of SEQ ID NO:2;        and    -   (c) the amino acid sequence encoded by the cDNA insert of clone        hGIL-19/AE289 deposited under accession number ATCC 207231;        the protein being substantially free from other mammalian        proteins. Preferably such protein comprises the amino acid        sequence of SEQ ID NO:2. In further preferred embodiments, the        present invention provides a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:2 having biological        activity, the fragment preferably comprising eight (more        preferably twenty, most preferably thirty) contiguous amino        acids of SEQ ID NO:2.

In certain preferred embodiments, the polynucleotide is operably linkedto an expression control sequence. The invention also provides a hostcell, including bacterial, yeast, insect and mammalian cells,transformed with such polynucleotide compositions. Also provided by thepresent invention are organisms that have enhanced, reduced, or modifiedexpression of the gene(s) corresponding to the polynucleotide sequencesdisclosed herein.

Processes are also provided for producing a protein, which comprise:

-   -   (a) growing a culture of the host cell transformed with such        polynucleotide compositions in a suitable culture medium; and    -   (b) purifying the protein from the culture.        The protein produced according to such methods is also provided        by the present invention.

Protein compositions of the present invention may further comprise apharmaceutically acceptable carrier. Compositions comprising an antibodywhich specifically reacts with such protein are also provided by thepresent invention.

Methods are also provided for preventing, treating or ameliorating amedical condition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising a proteinof the present invention and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid sequence of murine GIL-19 cDNA (SEQ IDNO:4).

DETAILED DESCRIPTION Isolated Proteins and Polynucleotides

Nucleotide and amino acid sequences, as presently determined, arereported below for each clone and protein disclosed in the presentapplication. The nucleotide sequence of each clone can readily bedetermined by sequencing of the deposited clone in accordance with knownmethods. The predicted amino acid sequence (both full-length and matureforms) can then be determined from such nucleotide sequence. The aminoacid sequence of the protein encoded by a particular clone can also bedetermined by expression of the clone in a suitable host cell,collecting the protein and determining its sequence.

As used herein a “secreted” protein is one which, when expressed in asuitable host cell, is transported across or through a membrane,including transport as a result of signal sequences in its amino acidsequence. “Secreted” proteins include without limitation proteinssecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. “Secreted” proteins alsoinclude without limitation proteins which are transported across themembrane of the endoplasmic reticulum.

Clone “hGIL-19/AE289”

A polynucleotide of the present invention has been identified initiallyas clone “hTIF/AE289”, later renamed and referred to herein also as“hGIL-19/AE289” and “hGIL-19”. Clone hGIL-19/AE289 was isolatedaccording to the following method. A murine EST was identified from amurine cDNA library made from splenocytes activated with both ConA andbone marrow derived dendritic cells. The EST was identified usingmethods which are selective for cDNAs encoding secreted proteins (seeU.S. Pat. No. 5,536,637). The murine EST sequence was used to isolate afull-length murine clone from the same cDNA library (SEQ ID NO:4; FIG. 1depicts the sequence of the murine GIL-19 cDNA). Analysis of thesequence of the murine clone revealed a significant homology tointerleukin-10 (IL-10).

In order to isolate a human homolog of the murine clone, PCR primerswere constructed based upon the region of the murine sequence whichshowed homology to IL-10. Use of such primers for amplification in ahuman PBMC library produced a PCR product of significant size. Analysisof the sequence of the PCR product confirmed that it was a homolog ofthe murine cDNA. Oligonucleotides were constructed from the sequence ofthe partial human clone and used to isolate a full-length human clonefrom the PBMC library.

hGIL-19/AE289 is a full-length human clone, including the entire codingsequence of a secreted protein (also referred to herein as “hTIF/AE289protein,” “hGIL-19/AE289 protein” and “hGIL-19 protein”). Analysis ofits sequence confirms its homology to IL-10.

The nucleotide sequence of hGIL-19 as presently determined is reportedin SEQ ID NO:1, and includes a poly(A) tail. The open reading frame andthe amino acid sequence of full-length hGIL-19 protein corresponding tothe foregoing nucleotide sequence is reported in SEQ ID NO:2. The aminoacid sequence of mature hGIL-19 corresponds to amino acids 34-179 of SEQID NO:2.

Clone “hGIL-19/AE289” was deposited on Apr. 28, 1999 with the AmericanType Culture Collection (10801 University Boulevard, Manassas, Va.20110-2209 U.S.A.) as an original deposit under the Budapest Treaty andwere given the accession number ATCC 207231. All restrictions on theavailability to the public of the deposited material will be irrevocablyremoved upon the granting of the patent, except for the requirementsspecified in 37 C.F.R. § 1.808(b), and the term of the deposit willcomply with 37 C.F.R. § 1.806.

Fragments of the proteins of the present invention (e.g. fragments whichare capable of exhibiting biological activity) are also encompassed bythe present invention. Fragments of the protein may be in linear form orthey may be cyclized using known methods, for example, as described inH. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. For example,fragments of the protein may be fused through “linker” sequences to theFc portion of an immunoglobulin. For a bivalent form of the protein,such a fusion could be to the Fc portion of an IgG molecule. Otherimmunoglobulin isotypes may also be used to generate such fusions. Forexample, a protein—IgM fusion would generate a decavalent form of theprotein of the invention.

The present invention also provides both full-length and mature forms ofthe disclosed proteins. The full-length form of the such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form(s) of such protein maybe obtained by expression of the disclosed full-length polynucleotide(preferably those deposited with ATCC) in a suitable mammalian cell orother host cell. The sequence(s) of the mature form(s) of the proteinmay also be determinable from the amino acid sequence of the full-lengthform and are set forth herein, for example as amino acids 1-179 of SEQID NO:2.

The present invention also provides genes corresponding to thepolynucleotide sequences disclosed herein. “Corresponding genes” are theregions of the genome that are transcribed to produce the mRNAs fromwhich cDNA polynucleotide sequences are derived and may includecontiguous regions of the genome necessary for the regulated expressionof such genes. Corresponding genes may therefore include but are notlimited to coding sequences, 5′ and 3′ untranslated regions,alternatively spliced exons, introns, promoters, enhancers, and silenceror suppressor elements. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials. An “isolated gene” is a gene that has beenseparated from the adjacent coding sequences, if any, present in thegenome of the organism from which the gene was isolated.

The chromosomal location corresponding to the polynucleotide sequencesdisclosed herein may also be determined, for example by hybridizingappropriately labeled polynucleotides of the present invention tochromosomes in situ. It may also be possible to determine thecorresponding chromosomal location for a disclosed polynucleotide byidentifying significantly similar nucleotide sequences in publicdatabases, such as expressed sequence tags (ESTs), that have alreadybeen mapped to particular chromosomal locations. For at least some ofthe polynucleotide sequences disclosed herein, public database sequenceshaving at least some similarity to the polynucleotide of the presentinvention have been listed by database accession number. Searches usingthe GenBank accession numbers of these public database sequences canthen be performed at an Internet site provided by the National Centerfor Biotechnology Information having the addresshttp://www.ncbi.nlm.nih.gov/UniGene/, in order to identify “UniGeneclusters” of overlapping sequences. Many of the “UniGene clusters” soidentified will already have been mapped to particular chromosomalsites.

Organisms that have enhanced, reduced, or modified expression of thegene(s) corresponding to the polynucleotide sequences disclosed hereinare provided. The desired change in gene expression can be achievedthrough the use of antisense polynucleotides or ribozymes that bindand/or cleave the mRNA transcribed from the gene (Albert and Morris,1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997,Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic AcidRes. Mol Biol 58: 1-39; all of which are incorporated by referenceherein). Transgenic animals that have multiple copies of the gene(s)corresponding to the polynucleotide sequences disclosed herein,preferably produced by transformation of cells with genetic constructsthat are stably maintained within the transformed cells and theirprogeny, are provided. Transgenic animals that have modified geneticcontrol regions that increase or reduce gene expression levels, or thatchange temporal or spatial patterns of gene expression, are alsoprovided (see European Patent No. 0 649 464 B1, incorporated byreference herein). In addition, organisms are provided in which thegene(s) corresponding to the polynucleotide sequences disclosed hereinhave been partially or completely inactivated, through insertion ofextraneous sequences into the corresponding gene(s) or through deletionof all or part of the corresponding gene(s). Partial or complete geneinactivation can be accomplished through insertion, preferably followedby imprecise excision, of transposable elements (Plasterk, 1992,Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2):719-722; all of which are incorporated by reference herein), or throughhomologous recombination, preferably detected by positive/negativegenetic selection strategies (Mansour et al., 1988, Nature 336: 348-352;U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396;5,616,491; and 5,679,523; all of which are incorporated by referenceherein). These organisms with altered gene expression are preferablyeukaryotes and more preferably are mammals. Such organisms are usefulfor the development of non-human models for the study of disordersinvolving the corresponding gene(s), and for the development of assaysystems for the identification of molecules that interact with theprotein product(s) of the corresponding gene(s).

Where the protein of the present invention is membrane-bound (e.g., is areceptor), the present invention also provides for soluble forms of suchprotein. In such forms, part or all of the intracellular andtransmembrane domains of the protein are deleted such that the proteinis fully secreted from the cell in which it is expressed. Theintracellular and transmembrane domains of proteins of the invention canbe identified in accordance with known techniques for determination ofsuch domains from sequence information. For example, the TopPredIIcomputer program can be used to predict the location of transmembranedomains in an amino acid sequence, domains which are described by thelocation of the center of the transmembrane domain, with at least tentransmembrane amino acids on each side of the reported centralresidue(s).

Proteins and protein fragments of the present invention include proteinswith amino acid sequence lengths that are at least 25% (more preferablyat least 50%, and most preferably at least 75%) of the length of adisclosed protein and have at least 60% sequence identity (morepreferably, at least 75% identity; most preferably at least 90% or 95%identity) with that disclosed protein, where sequence identity isdetermined by comparing the amino acid sequences of the proteins whenaligned so as to maximize overlap and identity while minimizing sequencegaps. Also included in the present invention are proteins and proteinfragments that contain a segment preferably comprising 8 or more (morepreferably 20 or more, most preferably 30 or more) contiguous aminoacids that shares at least 75% sequence identity (more preferably, atleast 85% identity; most preferably at least 95% identity) with any suchsegment of any of the disclosed proteins.

In another embodiment, proteins, protein fragments, and recombinantproteins of the present invention include those which can be identifiedbased on the presence of at least one “hGIL-19/AE289 receptor-bindingmotif.” As used herein, the term “HGIL-19/AE289 receptor-binding motif”includes amino acid sequences or residues which are important forbinding of hGIL-19 to its requisite receptor. In a preferred embodiment,a hGIL-19 protein contains a hGIL-19/AE289 receptor-binding motifincluding about amino acids 50-60 of SEQ ID NO:2. In another embodiment,a GIL-19 protein contains a hGIL-19/AE289 receptor-binding motifincluding about amino acids 63-81 of SEQ ID NO:2. In yet anotherembodiment, a GIL-19 protein contains a hGIL-19/AE289 receptor-bindingmotif including about amino acids 168-177 of SEQ ID NO:2. In a preferredembodiment, a GIL-19 protein contains a hGIL-19/AE289 receptor-bindingmotif including at least one of amino acids 50-60, amino acids 63-81,and/or about amino acids 168-177 of SEQ ID NO:2.

In yet another embodiment, a hGIL-19/AE289 receptor binding motif has anamino acid sequence at least 95%, 96%, 97%, 98%, 99%, or more identicalto an amino acid sequence selected from the group consisting of aminoacids 50-60 of SEQ ID NO:2, amino acids 63-81 of SEQ ID NO:2, and aminoacids 168-177 of SEQ ID NO:2.

In another embodiment, proteins, protein fragments, and recombinantproteins of the present invention include those which can be identifiedbased on the presence of at least one, two, three, four or more sitesfor N-linked glycosylation.

In particular, sequence identity may be determined using WU-BLAST(Washington University BLAST) version 2.0 software, which builds uponWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignmentstatistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschulet al., 1990, Basic local alignment search tool, Journal of MolecularBiology 215: 403-410; Gish and States, 1993, Identification of proteincoding regions by database similarity search, Nature Genetics 3:266-272; Karlin and Altschul, 1993, Applications and statistics formultiple high-scoring segments in molecular sequences, Proc. Natl. Acad.Sci. USA 90: 5873-5877; all of which are incorporated by referenceherein). WU-BLAST version 2.0 executable programs for several UNIXplatforms can be downloaded fromftp://blast.wustl.edu/blast/executables. The complete suite of searchprograms (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided atthat site, in addition to several support programs. WU-BLAST 2.0 iscopyrighted and may not be sold or redistributed in any form or mannerwithout the express written consent of the author; but the postedexecutables may otherwise be freely used for commercial, nonprofit, oracademic purposes. In all search programs in the suite—BLASTP, BLASTN,BLASTX, TBLASTN and TBLASTX—the gapped alignment routines are integralto the database search itself, and thus yield much better sensitivityand selectivity while producing the more easily interpreted output.Gapping can optionally be turned off in all of these programs, ifdesired. The default penalty (Q) for a gap of length one is Q=9 forproteins and BLASTP, and Q=10 for BLASTN, but may be changed to anyinteger value including zero, one through eight, nine, ten, eleven,twelve through twenty, twenty-one through fifty, fifty-one through onehundred, etc. The default per-residue penalty for extending a gap (R) isR=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed toany integer value including zero, one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve through twenty, twenty-onethrough fifty, fifty-one through one hundred, etc. Any combination ofvalues for Q and R can be used in order to align sequences so as tomaximize overlap and identity while minimizing sequence gaps. Thedefault amino acid comparison matrix is BLOSUM62, but other amino acidcomparison matrices such as PAM can be utilized.

Species homologues of the disclosed polynucleotides and proteins arealso provided by the present invention. As used herein, a “specieshomologue” is a protein or polynucleotide with a different species oforigin from that of a given protein or polynucleotide, but withsignificant sequence similarity to the given protein or polynucleotide.Preferably, polynucleotide species homologues have at least 60% sequenceidentity (more preferably, at least 75%, 80%, 85%, 90%, 95%, 99%) withthe given polynucleotide, and protein species homologues have at least30% sequence identity (more preferably, at least 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%) with the given protein, where sequenceidentity is determined by comparing the nucleotide sequences of thepolynucleotides or the amino acid sequences of the proteins when alignedso as to maximize overlap and identity while minimizing sequence gaps.Species homologues may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source from the desired species. Preferably,species homologues are those isolated from mammalian species. Mostpreferably, species homologues are those isolated from certain mammalianspecies such as, for example, Pan troglodytes, Gorilla gorilla, Pongopygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papiohamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus,Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus,Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris,Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equuscaballus, for which genetic maps have been created allowing theidentification of syntenic relationships between the genomicorganization of genes in one species and the genomic organization of therelated genes in another species (O'Brien and Seuanez, 1988, Ann. Rev.Genet. 22: 323-351; O'Brien et al, 1993, Nature Genetics 3:103-112;Johansson et al., 1995, Genomics 25: 682-690; Lyons et al., 1997, NatureGenetics 15: 47-56; O'Brien et al., 1997, Trends in Genetics 13(10):393-399; Carver and Stubbs, 1997, Genome Research 7:1123-1137; all ofwhich are incorporated by reference herein).

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotides which also encode proteins whichare identical or have significantly similar sequences to those encodedby the disclosed polynucleotides. Preferably, allelic variants have atleast 60% sequence identity (more preferably, at least 75%, 80%, 85%,90%, 95%, 99%) with the given polynucleotide, where sequence identity isdetermined by comparing the nucleotide sequences of the polynucleotideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. Allelic variants may be isolated and identified by makingsuitable probes or primers from the sequences provided herein andscreening a suitable nucleic acid source from individuals of theappropriate species.

The invention also includes polynucleotides with sequences complementaryto those of the polynucleotides disclosed herein.

The present invention also includes polynucleotides that hybridize underreduced stringency conditions, more preferably stringent conditions, andmost preferably highly stringent conditions, to polynucleotidesdescribed herein. Examples of stringency conditions are shown in thetable below: highly stringent conditions are those that are at least asstringent as, for example, conditions A-F; stringent conditions are atleast as stringent as, for example, conditions G-L; and reducedstringency conditions are at least as stringent as, for example,conditions M-R.

Hybrid Wash Stringency Polynucleotide Length Hybridization Temperatureand Temperature and Condition Hybrid (bp)^(‡) Buffer^(†) Buffer^(†) ADNA:DNA ≧50 65° C.; 1xSSC -or- 65° C.; 0.3xSSC 42° C.; 1xSSC, 50%formamide B DNA:DNA <50 T_(B)*; 1xSSC T_(B)*; 1xSSC C DNA:RNA ≧50 67°C.; 1xSSC -or- 67° C.; 0.3xSSC 45° C.; 1xSSC, 50% formamide D DNA:RNA<50 T_(D)*; 1xSSC T_(D)*; 1xSSC E RNA:RNA ≧50 70° C.; 1xSSC -or- 70° C.;0.3xSSC 50° C.; 1xSSC, 50% formamide F RNA:RNA <50 T_(F)*; 1xSSC T_(F)*;1xSSC G DNA:DNA ≧50 65° C.; 4xSSC -or- 65° C.; 1xSSC 42° C.; 4xSSC, 50%formamide H DNA:DNA <50 T_(H)*; 4xSSC T_(H)*; 4xSSC I DNA:RNA ≧50 67°C.; 4xSSC -or- 67° C.; 1xSSC 45° C.; 4xSSC, 50% formamide J DNA:RNA <50T_(J)*; 4xSSC T_(J)*; 4xSSC K RNA:RNA ≧50 70° C.; 4xSSC -or- 67° C.;1xSSC 50° C.; 4xSSC, 50% formamide L RNA:RNA <50 T_(L)*; 2xSSC T_(L)*;2xSSC M DNA:DNA ≧50 50° C.; 4xSSC -or- 50° C.; 2xSSC 40° C.; 6xSSC, 50%formamide N DNA:DNA <50 T_(N)*; 6xSSC T_(N)*; 6xSSC O DNA:RNA ≧50 55°C.; 4xSSC -or- 55° C.; 2xSSC 42° C.; 6xSSC, 50% formamide P DNA:RNA <50T_(P)*; 6xSSC T_(P)*; 6xSSC Q RNA:RNA ≧50 60° C.; 4xSSC -or- 60° C.;2xSSC 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 T_(R)*; 4xSSC T_(R)*;4xSSC ^(‡)The hybrid length is that anticipated for the hybridizedregion(s) of the hybridizing polynucleotides. When hybridizing apolynucleotide to a target polynucleotide of unknown sequence, thehybrid length is assumed to be that of the hybridizing polynucleotide.When polynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.^(†)SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4)can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes after hybridization is complete. *T_(B)-T_(R): Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m)(° C.) = 2(# of A + T bases) + 4(# of G + C bases). Forhybrids between 18 and 49 base pairs in length, T_(m)(° C.) = 81.5 +16.6(log₁₀ [Na⁺]) + 0.41(% G + C) − (600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1xSSC = 0.165 M).

Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

Preferably, each such hybridizing polynucleotide has a length that is atleast 25% (more preferably at least 50%, and most preferably at least75%) of the length of the polynucleotide of the present invention towhich it hybridizes, and has at least 60% sequence identity (morepreferably, at least 75% identity; most preferably at least 90% or 95%identity) with the polynucleotide of the present invention to which ithybridizes, where sequence identity is determined by comparing thesequences of the hybridizing polynucleotides when aligned so as tomaximize overlap and identity while minimizing sequence gaps.

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 44854490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculoviruslinsect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,Heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLabs (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and Invitrogen Corporation (Carlsbad, Calif.),respectively. The protein can also be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. One such epitope (“Flag”) is commercially available from theEastman Kodak Company (New Haven, Conn.).

In addition, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are believed to be encompassed by the presentinvention.

Uses and Biological Activity

The polynucleotides and proteins of the present invention can exhibitone or more of the uses or biological activities (including thoseassociated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

hgL-19 Uses

Because of its homology to IL-10, human GIL-19/AE289 can be considered amember of the general family of cytokines and, as such, can exhibitsimilar activities to IL-10. Cytokines play important roles both inhealth and disease and have multiple clinical indications. Thereforethis molecule (and other molecules of the present invention) will beuseful as an agonist in certain clinical indications and antagonists ofthis molecule will be useful in other clinical situations, particularlyin those in which IL-10 acts as an agonist or IL-10 antagonists act asan antagonist. Whether the agonist or antagonist is the preferred drugwill depend on the particular aspects of the disease pathology, such asthe cell types involved, the nature of the stimulus and the cellularmicroenvironment.

In a preferred embodiment, a HGIL-19 activity is at least one or more ofthe following activities: (1) modulating, for example antagonizing asignal transduction pathway (e.g. a GIL-19 dependent pathway); (2)modulating cytokine production and/or secretion (e.g. production and/orsecretion of a proinflammatory cytokine); (3) modulating lymphokineproduction and/or secretion; (4) modulating production of adhesionmolecules and/or cellular adhesion; (5) modulating expression oractivity of nuclear transcription factors; (7) modulating secretion ofIL-1; (8) competing with receptors for other cytokines; (9) competingwith another hGIL-19 family member protein to bind a hGIL-19 receptor;(10) modulating nuclear translocation of internalized receptor forhGIL-19 or another cytokine or ligand-complexed receptor; (11)modulating cell proliferation, development or differentiation, forexample, cytokine-stimulated or a hGIL-19 protein-stimulatedproliferation, development or differentiation (e.g., of an epithelialcell, for example, a squamous epithelial cell of the esophagus, or of askin cell, e.g., a keratinocyte); (12) modulating cell proliferation,development or differentiation of an osteogenic cell (e.g., of anosteoclast precursor cell, osteoclast and/or osteoblast); (13)modulating bone formation, bone metabolism and/or bone homeostasis(e.g., inhibiting bone resorption); (15) modulating cellular immuneresponses; (16) modulating cytokine-mediated proinflammatory actions(e.g., inhibiting acute phase protein synthesis by hepatocytes, fever,and/or prostaglandin synthesis, for example PGE₂ synthesis); and (17)promoting and/or potentiating wound healing.

Considering its apparent immunomodulatory role, human GIL-19/AE289proteins may act on the following cell types: T cells, B cells,dendritic cells, macrophages/monocytes, neutrophils, mast cells,basophils, eosinophils, antigen presenting cells of the nervous systemand antigen presenting cells of the kidney. Based on its homology toIL-10, human GIL-19/AE289 proteins (or agonists or antagonists thereof)can have the following activities and uses:

-   -   (a) Upregulation of humoral immune responses and attenuates cell        mediated immune reactions;    -   (b) Function as an anti-inflammatory agent by inhibiting the        synthesis of pro-inflammatory cytokines and chemokines;    -   (c) Modulation of inflammatory responses associated with injury,        sepsis, gastrointestinal and cardiovascular disease, and        inflammation following surgery;    -   (d) Treatment of acute myelogenous leukemia, Non-Hodgkin's        lymphoma, bone marrow transplantation to treat recipient before        engraftment, bone marrow transplantation to treat stem cells of        donor before transplantation, and to ameliorate graft versus        host disease following bone marrow transplantation;    -   (e) Treatment cell mediated autoimmune diseases such as multiple        sclerosis, diabetes, rheumatoid arthritis, myasthenia gravis,        systemic lupus erythematosus, nephrotoxicity associated with        glomerulonephritis, inflammatory bowel disease, Crohn's disease,        pancreatitis, and asthma.

Human GIL-19/AE289 agonists include without limitation humanGIL-19/AE289 proteins and fragments, deletion mutants and additionmutants thereof; and peptide and small molecule compounds that interactwith the receptor or other target to which human GIL-19/AE289 isdirected. Human GIL-19/AE289 antagonists include without limitationantibodies directed to human GIL-19/AE289 proteins; soluble forms of thereceptor or other target to which human GIL-19/AE289 is directed;antibodies directed to the receptor or other target to which humanGIL-19/AE289 is directed; and peptide and small molecule compounds thatinhibit or interfere with the interaction of human GIL-19/AE289 with itsreceptor or other target.

Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on Southern gels; as chromosomemarkers or tags (when labeled) to identify chromosomes or to map relatedgene positions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, thosedescribed in Gyuris et al., 1993, Cell 75: 791-803 and in Rossi et al.,1997, Proc. Natl. Acad. Sci. USA 94: 8405-8410, all of which areincorporated by reference herein) to identify polynucleotides encodingthe other protein with which binding occurs or to identify inhibitors ofthe binding interaction.

The proteins provided by the present invention can similarly be used inassays to determine biological activity, including in a panel ofmultiple proteins for high-throughput screening; to raise antibodies orto elicit another immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its receptor) in biological fluids; as markers for tissuesin which the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state); and, of course, to isolatecorrelative receptors or ligands. Where the protein binds or potentiallybinds to another protein (such as, for example, in a receptor-ligandinteraction), the protein can be used to identify the other protein withwhich binding occurs or to identify inhibitors of the bindinginteraction. Proteins involved in these binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor-dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123,T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of theinvention may, among other means, be measured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose

described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. GreenePublishing Associates and Wiley-Interscience (Chapter 3, In Vitro assaysfor Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studiesin Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolliet al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol.149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. Coligan eds.Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human Interferon γ, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, JohnWiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S, and Lipsky, P. E. In Current Protocols inImmunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6-Nordan, R. In Current Protocols in Immunology. J. E.e. a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e. a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e. a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byviral (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoimmune disorders. More specifically, infectious diseasescauses by viral, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malariaspp. and various fungal infections such as candidiasis. Of course, inthis regard, a protein of the present invention may also be useful wherea boost to the immune system generally may be desirable, i.e., in thetreatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

Using the proteins of the invention it may also be possible to regulateimmune responses in a number of ways. Down regulation may be in the formof inhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafis in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci. USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

In another application, up regulation or enhancement of antigen function(preferably B lymphocyte antigen function) may be useful in theinduction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. For example, tumor cells obtained from apatient can be transfected ex vivo with an expression vector directingthe expression of a peptide having B7-2-like activity alone, or inconjunction with a peptide having B7-1-like activity and/or B7-3-likeactivity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a T cellmediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient amounts of MHC class I or MHCclass II molecules, can be transfected with nucleic acid encoding all ora portion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I α chain protein and β₂ microglobulin protein or an MHC class IIα chain protein and an MHC class II β chain protein to thereby expressMHC class I or MHC class II proteins on the cell surface. Expression ofthe appropriate class I or class 11 MHC in conjunction with a peptidehaving the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3)induces a T cell mediated immune response against the transfected tumorcell. Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, WStrober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowman et al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide an environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate. A protein of the invention may also exhibit angiogenicactivity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

A protein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H Iand Rovee, D T, eds.), Year Book Medical Publishers, Inc., Chicago, asmodified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins and are characterized by their ability to stimulate the releaseof follicle stimulating hormone (FSH). Thus, a protein of the presentinvention, alone or in heterodimers with a member of the inhibin afamily, may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in these mammals. Alternatively,the protein of the invention, as a homodimer or as a heterodimer withother protein subunits of the inhibin-β group, may be useful as afertility inducing therapeutic, based upon the ability of activinmolecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokineticactivity (e.g., act as a chemokine) for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. Chemotactic andchemokinetic proteins can be used to mobilize or attract a desired cellpopulation to a desired site of action. Chemotactic or chemokineticproteins provide particular advantages in treatment of wounds and othertrauma to tissues, as well as in treatment of localized infections. Forexample, attraction of lymphocytes, monocytes or neutrophils to tumorsor sites of infection may result in improved immune responses againstthe tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994;Johnston et al. J. of Immunol. 153: 1762-1768, 1994.

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. As a result, such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. Examples of such receptors and ligands include, withoutlimitation, cytokine receptors and their ligands, receptor kinases andtheir ligands, receptor phosphatases and their ligands, receptorsinvolved in cell-cell interactions and their ligands (including withoutlimitation, cellular adhesion molecules (such as selectins, integrinsand their ligands) and receptor/ligand pairs involved in antigenpresentation, antigen recognition and development of cellular andhumoral immune responses). Receptors and ligands are also useful forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction. A protein of the present invention(including, without limitation, fragments of receptors and ligands) maythemselves be useful as inhibitors of receptor/ligand interactions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

Anti-Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationinflammation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material.

Cadherin/Tumor Invasion Suppressor Activity

Cadherins are calcium-dependent adhesion molecules that appear to playmajor roles during development, particularly in defining specific celltypes. Loss or alteration of normal cadherin expression can lead tochanges in cell adhesion properties linked to tumor growth andmetastasis. Cadherin malfunction is also implicated in other humandiseases, such as pemphigus vulgaris and pemphigus foliaceus(auto-immune blistering skin diseases), Crohn's disease, and somedevelopmental abnormalities.

The cadherin superfamily includes well over forty members, each with adistinct pattern of expression. All members of the superfamily have incommon conserved extracellular repeats (cadherin domains), butstructural differences are found in other parts of the molecule. Thecadherin domains bind calcium to form their tertiary structure and thuscalcium is required to mediate their adhesion. Only a few amino acids inthe first cadherin domain provide the basis for homophilic adhesion;modification of this recognition site can change the specificity of acadherin so that instead of recognizing only itself, the mutant moleculecan now also bind to a different cadherin. In addition, some cadherinsengage in heterophilic adhesion with other cadherins.

E-cadherin, one member of the cadherin superfamily, is expressed inepithelial cell types. Pathologically, if E-cadherin expression is lostin a tumor, the malignant cells become invasive and the cancermetastasizes. Transfection of cancer cell lines with polynucleotidesexpressing E-cadherin has reversed cancer-associated changes byreturning altered cell shapes to normal, restoring cells' adhesivenessto each other and to their substrate, decreasing the cell growth rate,and drastically reducing anchorage-independent cell growth. Thus,reintroducing E-cadherin expression reverts carcinomas to a lessadvanced stage. It is likely that other cadherins have the same invasionsuppressor role in carcinomas derived from other tissue types.Therefore, proteins of the present invention with cadherin activity, andpolynucleotides of the present invention encoding such proteins, can beused to treat cancer. Introducing such proteins or polynucleotides intocancer cells can reduce or eliminate the cancerous changes observed inthese cells by providing normal cadherin expression.

Cancer cells have also been shown to express cadherins of a differenttissue type than their origin, thus allowing these cells to invade andmetastasize in a different tissue in the body. Proteins of the presentinvention with cadherin activity, and polynucleotides of the presentinvention encoding such proteins, can be substituted in these cells forthe inappropriately expressed cadherins, restoring normal cell adhesiveproperties and reducing or eliminating the tendency of the cells tometastasize.

Additionally, proteins of the present invention with cadherin activity,and polynucleotides of the present invention encoding such proteins, canused to generate antibodies recognizing and binding to cadherins. Suchantibodies can be used to block the adhesion of inappropriatelyexpressed tumor-cell cadherins, preventing the cells from forming atumor elsewhere. Such an anti-cadherin antibody can also be used as amarker for the grade, pathological type, and prognosis of a cancer, i.e.the more progressed the cancer, the less cadherin expression there willbe, and this decrease in cadherin expression can be detected by the useof a cadherin-binding antibody.

Fragments of proteins of the present invention with cadherin activity,preferably a polypeptide comprising a decapeptide of the cadherinrecognition site, and polynucleotides of the present invention encodingsuch protein fragments, can also be used to block cadherin function bybinding to cadherins and preventing them from binding in ways thatproduce undesirable effects. Additionally, fragments of proteins of thepresent invention with cadherin activity, preferably truncated solublecadherin fragments which have been found to be stable in the circulationof cancer patients, and polynucleotides encoding such protein fragments,can be used to disturb proper cell-cell adhesion.

Assays for cadherin adhesive and invasive suppressor activity include,without limitation, those described in: Hortsch et al. J Biol Chem 270(32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995;Ozawa et al. Cell 63: 1033-1038, 1990.

Tumor Inhibition Activity

In addition to the activities described above for immunologicaltreatment or prevention of tumors, a protein of the invention mayexhibit other anti-tumor activities. A protein may inhibit tumor growthdirectly or indirectly (such as, for example, via antibody-dependentcell-mediated cytotoxicity (ADCC)). A protein may exhibit its tumorinhibitory activity by acting on tumor tissue or tumor precursor tissue,by inhibiting formation of tissues necessary to support tumor growth(such as, for example, by inhibiting angiogenesis), by causingproduction of other factors, agents or cell types which inhibit tumorgrowth, or by suppressing, eliminating or inhibiting factors, agents orcell types which promote tumor growth.

Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or caricadic cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

Administration and Dosing

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be used in a pharmaceutical composition when combined witha pharmaceutically acceptable carrier. Such a composition may alsocontain (in addition to protein and a carrier) diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other agents which either enhance theactivity of the protein or compliment its activity or use in treatment.Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with proteinof the invention, or to minimize side effects. Conversely, protein ofthe present invention may be included in formulations of the particularcytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent to minimize sideeffects of the cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

A protein of the present invention may be active in multimers (e.g.,heterodimers or homodimers) or complexes with itself or other proteins.As a result, pharmaceutical compositions of the invention may comprise aprotein of the invention in such multimeric or complexed form.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunolgobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

Administration of protein of the present invention used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

When a therapeutically effective amount of protein of the presentinvention is administered orally, protein of the present invention willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein of the present invention, and preferably from about 1to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 ng to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the protein of the present inventionwill be in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. As used herein, the term “antibody” includes without limitationa polyclonal antibody, a monoclonal antibody, a chimeric antibody, asingle-chain antibody, a CDR-grafted antibody, a humanized antibody, orfragments thereof which bind to the indicated protein. Such term alsoincludes any other species derived from an antibody or antibody sequencewhich is capable of binding the indicated protein.

Antibodies to a particular protein can be produced by methods well knownto those skilled in the art. For example, monoclonal antibodies can beproduced by generation of antibody-producing hybridomas in accordancewith known methods (see for example, Goding, 1983, Monoclonalantibodies: principles and practice, Academic Press Inc., New York; andYokoyama, 1992, “Production of Monoclonal Antibodies” in CurrentProtocols in Immunology, Unit 2.5, Greene Publishing Assoc. and JohnWiley & Sons). Polyclonal sera and antibodies can be produced byinoculation of a mammalian subject with the relevant protein orfragments thereof in accordance with known methods. Fragments ofantibodies, receptors, or other reactive peptides can be produced fromthe corresponding antibodies by cleavage of and collection of thedesired fragments in accordance with known methods (see for example,Goding, supra; and Andrew et al., 1992, “Fragmentation ofImmunoglobulins” in Current Protocols in Immunology, Unit 2.8, GreenePublishing Assoc. and John Wiley & Sons). Chimeric antibodies and singlechain antibodies can also be produced in accordance with knownrecombinant methods (see for example, U.S. Pat. Nos. 5,169,939,5,194,594, and 5,576,184). Humanized antibodies can also be made fromcorresponding murine antibodies in accordance with well known methods(see for example, U.S. Pat. Nos. 5,530,101, 5,585,089, and 5,693,762).Additionally, human antibodies may be produced in non-human animals suchas mice that have been genetically altered to express human antibodymolecules (see for example Fishwild et al., 1996, Nature Biotechnology14: 845-851; Mendez et al., 1997, Nature Genetics 15: 146-156 (erratumNature Genetics 16: 410); and U.S. Pat. Nos. 5,877,397 and 5,625,126).Such antibodies may be obtained using either the entire protein orfragments thereof as an immunogen. The peptide immunogens additionallymay contain a cysteine residue at the carboxyl terminus, and areconjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methodsfor synthesizing such peptides are known in the art, for example, as inR. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L.Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to the protein of the invention may beuseful diagnostic agents for the immunodetection of the protein.Neutralizing monoclonal antibodies binding to the protein may also beuseful therapeutics for both conditions associated with the protein andalso in the treatment of some forms of cancer where abnormal expressionof the protein is involved. In the case of cancerous cells or leukemiccells, neutralizing monoclonal antibodies against the protein may beuseful in detecting and preventing the metastatic spread of thecancerous cells, which may be mediated by the protein.

For compositions of the present invention which are useful for bone,cartilage, tendon or ligament regeneration, the therapeutic methodincludes administering the composition topically, systematically, orlocally as an implant or device. When administered, the therapeuticcomposition for use in this invention is, of course, in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablybe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than a protein of the invention which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing composition to the site of bone and/orcartilage damage, providing a structure for the developing bone andcartilage and optimally capable of being resorbed into the body. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics. Matrices may be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics may be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability.

Presently preferred is a 50:50 (mole weight) copolymer of lactic acidand glycolic acid in the form of porous particles having diametersranging from 150 to 800 microns. In some applications, it will be usefulto utilize a sequestering agent, such as carboxymethyl cellulose orautologous blood clot, to prevent the protein compositions fromdisassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells.

In further compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention.

The dosage regimen of a protein-containing pharmaceutical composition tobe used in tissue regeneration will be determined by the attendingphysician considering various factors which modify the action of theproteins, e.g., amount of tissue weight desired to be formed, the siteof damage, the condition of the damaged tissue, the size of a wound,type of damaged tissue (e.g., bone), the patient's age, sex, and diet,the severity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor 1), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA).

Cells may also be cultured ex vivo in the presence of proteins of thepresent invention in order to proliferate or to produce a desired effecton or activity in such cells. Treated cells can then be introduced invivo for therapeutic purposes.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Sequence Listing, are incorporated herein byreference.

EXAMPLES Identification and Characterization of Clone “hGIL-19/AE289”

A polynucleotide of the present invention has been identified as clone“hGIL-19/AE289”. Clone hGIL-19/AE289 was isolated according to thefollowing method. A murine EST was identified from a murine cDNA librarymade from splenocytes activated with both ConA and bone marrow deriveddendritic cells. The EST was identified using methods which areselective for cDNAs encoding secreted proteins (see U.S. Pat. No.5,536,637). The murine EST sequence was used to isolate a full-lengthmurine clone from the same cDNA library. Analysis of the sequence of themurine clone revealed a significant homology to interleukin-10 (IL-10).

In order to isolate a human homolog of the murine clone, PCR primerswere constructed based upon the region of the murine sequence whichshowed homology to IL-10. Use of such primers for amplification in ahuman PBMC library produced a PCR product of significant size. Analysisof the sequence of the PCR product confirmed that it was a homolog ofthe murine cDNA. Oligonucleotides were constructed from the sequence ofthe partial human clone and used to isolate a full-length human clonefrom the PBMC library.

hGIL-19/AE289 is a full-length human clone, including the entire codingsequence of a secreted protein (also referred to herein as“hGIL-19/AE289” protein). Analysis of its amino acid sequence indicatedthat it has about 23% homology to hIL-10. Based on the putativereceptor-binding motifs in IL-10, three motifs involved with analogousfunction have been proposed in hGIL-19/AE289 through computer modeling.These are the regions of SEQ ID NO:2 from residue 50 to 60, from residue63 to 81, and from residue 168 to 177. Analyses of databases revealedthat hGIL-19 also exhibits similar levels of homology with IL-10 ofother species.

The nucleotide sequence of hGIL-19/AE289 as presently determined isreported in SEQ ID NO: 1, and includes a poly(A) tail. The amino acidsequence of the hGIL-19/AE289 protein corresponding to the foregoingnucleotide sequence is reported in SEQ ID NO:2.

Characterization of hGIL-19/AE289 Protein Cell lines which stablyexpress and secrete full length hGIL-19/AE289 protein were created bytransfecting CHO cells with hGIL-19/AE289 cDNA in appropriate expressionvectors. Transiently transfected COS cells using appropriatehGIL-19/AE289 expression vectors have been used to make hGIL-19/AE289protein for analysis. Transfections were accomplished using thecommercially available Lipofectamine reagent (Gibco). Interestingly, COScells which express hGIL-19 were observed to non-uniformly detach,forming holes in the cell culture monolayer. Media conditioned bytransfected COS cells was used to demonstrate cytokine-like activity ofhGIL-19/AE289 protein. Western blot analysis of cell lysates showed thatStat-3 becomes phosphorylated (activated) in a kidney mesangialtissue-derived cell line exhibiting macrophage-like qualities (MES-13;see, Durnoutier et al (2000) J. of Immunology 164:1814-1819) uponexposure of that cell to media conditioned by hGIL-19/AE289-expressingcells. In addition phosphorylation of Stat-3 is induced innon-transfected COS cells that are treated with hGIL-19 protein.

Electrophoretic analysis of hGIL-19/AE289 protein (derived from thetransfected COS cell lines described herein) indicated that theexpressed protein exists in a range of sizes. Treatment of COS-derivedhGIL-19 protein with N-glycanase prior to electrophoresis results in asingle band corresponding to the highest mobility (e.g. lowest molecularweight) species seen in untreated hGIL-19/AE289. This is consistent withproposed glycosylation events which may occur at the putative N-linkedglycosylation sites identified in the amino acid sequence ofhGIL-19/AE289 (amino acid residues 54-56, 68-70, 97-99, and 176-178 ofSEQ ID NO:2).

Edman N-terminal sequencing determined that the N-terminus of the maturehGIL-19/AE289 protein begins with the residue at position 34 of SEQ IDNO:2 (alanine). Expression vectors were created which fuse a “6xhistidine” affinity tag and a FLAG epitope tag to the N-terminus of themature hGIL-19/AE289 protein. (The added amino acid tag is given in SEQID NO:3 and has the following amino acid sequence:MKFLVNVALVFMVVYISYIYAGSGHHHHHHGSGDYKDDDDKAPISSHCR). These taggedconstructs were used to create stably expressing CHO cell lines andtransiently expressing COS cell lines. The tags provided a convenientmeans for detecting hGIL-19/AE289(e.g., anti-6xhis antibodies; anti-FLAGantibodies), and for purifying the protein from conditioned media (usingNi⁺² resin). Human GIL-19 protein purified by this tag from thehGIL-19/AE289-expressing COS cell lines could used to induce Stat-3activationin MES-13 cells.

Comparison of hGIL-19 mRNA transcripts in activated Th1 and Th2 cells(see, for example, Syrbe et al, (1999)Springer Seminars inImmunopathology, 21:263-85) indicated a substantially higher level ofexpression of GIL-19 in activated Th1 cells than in activated Th2 cells.Analysis of GIL-19 mRNA was accomplished with RNAse protection assays.

Immunological Effects GIL-19

The immunological effects of GIL-19 were investigated in a metazoancontext by viral introduction of the cDNA of murine GIL-19 into mice. Anadenoviral vector was used to express a cDNA of murine GIL-19 in 8 weekold C57/B6 female mice by injection of 5×10¹⁰ viral particles. Test micewere sacrificed at 7 and 14 days after injection and compared withcontrol mice injected with buffer only or with adenovirus expressinggreen fluorescent protein (GFP). At days 7 and 14, it was noted that theabsolute and relative thymic weights were significantly decreased in themice which expressed the viral murine GIL-19. Absolute mean weight ofthe spleen was decreased on day 14 and liver weights were slightlyincreased on day 7. A gross generalized atrophy of the thymus as well aslymphoid depletion (observed microscopically) was apparent on days 7 and14.

In addition, there were a number of hematological effects that wereapparent on day 7, including decreased red blood cell count, hemoglobin,and hematocrit. These effects, taken together, indicated anemia in theanimals. Furthermore, there was an increase in platelets as well as anincrease in the white blood cell count due to an increase ofneutrophils. In light of these observations there was no evidence of aregenerative response, which indicated that the effects may be at thelevel of the bone marrow.

Furthermore, there was a slight decrease in Albumin levels at day 7 andday 14. A possible cause for this is the loss of small molecules throughthe kidney or gut.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated protein comprising an amino acid sequence selected fromthe group consisting of: (a) the amino acid sequence of SEQ ID NO:2; (b)a fragment of the amino acid sequence of SEQ ID NO:2, the fragmentcomprising thirty contiguous amino acids of SEQ ID NO:2; and (c) theamino acid sequence encoded by the cDNA insert of clone hGIL-19/AE289deposited under accession number ATCC 207231; the protein beingsubstantially free from other mammalian proteins.